Monoclonal antibody specifically binding to human plasmalemma vesicle-associated protein pv-1, preparation and use thereof

ABSTRACT

Disclosed herein is a monoclonal antibody or a derivative thereof that specifically hinds to human plasmalemma vesicle-associated protein (PLVAP, PV-1), including antigen complementarity-determining regions CDR1, CDR2 and CDR3 of an antibody light chain variable region, and antigen complementarity-determining regions CDR1, CDR2 and CDR3 of an antibody heavy chain variable region. The invention also provides a preparation process of a human-mouse chimeric antibody and amino acid sequences of the antibody heavy chain variable region and the antibody light chain variable region. The monoclonal antibody or derivative thereof can be used as a component of a pharmaceutical composition or prepared into a suitable medicament, administered alone or combined with other medications such as anti-VEGF monoclonal antibody and the like, for treating choroidal neovascularization fundus diseases and other angiogenesis/osmosis-related diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national stage entry of InternationalApplication No. PCT /CN2018/107809, filed on Sep. 27, 2018, which isbased upon and claims priority to Chinese Patent Application No.201810266295.5, filed on Mar. 30, 2018, the entire contents of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy is named GBSHHY010-SequenceListing-20200807.txt, created on Aug. 7, 2020 and is 22 KB in size.

TECHNICAL FIELD

The present invention belongs to the field of biotechnology involvingmonoclonal antibody. The present invention relates to a monoclonalantibody specifically binding to human plasmalemma vesicle-associatedprotein (PLVAP, PV-1 for short) and its coding sequences, as well aspreparation and use thereof.

BACKGROUND

In the human body, the vascular system, constructed with endothelialcells (EC) lining in the innermost layer of blood vessels in variousorgans and tissues, the surrounding pericyte, and basement plays thefollowing dual and complementary roles:

1) Separating blood circulating in the vessel wall from the tissueoutside of the wall, thus act as a physical barrier.

2) Mediating the exchange of O₂, CO₂, H₂O, and electrolytes,transportation of hormone/protein or other nutrients and metabolites,migration of inflammatory cells/immune cells, etc., thus playing apermeability role.

In some tissues or organs which are highly active in blood-tissuesubstance exchange and metabolisms, such as endocrine glands, liverblood sinuses, glomeruli, bone marrow, spleen, gastrointestinalepithelium, brain, and retina plexus of the eye, the vascularendothelium is not entirely continuous or surrounded by pericyte, butappears discontinuous or sinusoid (see review: Crivellato E, Nico B andRibatti D, 2007 Contribution of endothelial cells to organogenesis, amodern reappraisal of an old Aristotelian concept. J. Anat 211:415-427).The surface of vascular endothelium or the wall of the vessel in theseareas has typically many fenestrae or caveolae (also known asplasmalemmal vesicle) structure with a diameter of around 60-80 nm. Thefenestrae often cluster orderly and equidistantly in dozens or hundreds,which can take on the shape of a sieve plate or honeycomb under theelectron microscope. The fenestral diaphragm structure, which is onlyabout 6-7 nm thick, is embedded in the interior of some fenestrae(Bearer E L and Orci L. 1985 J Cell Biol. 100:418-428 ; Peters K R,Carley W W, Palade G E. 1985 J Cell Biol. 101;2233-8; Lomardi Tet al.,1986 J Cell Biol. 102: 1965-1970).

There usually are two routes of substance exchange and cells migrationin blood-tissue: 1) para-cellular migration through the space betweenthe endothelial cells of a blood vessel; 2) trans-endothelial migrationfrom one side of the vascular wall to the other through fenestrae orcaveolae in vascular endothelium/wall of the vessel. Some factors, suchas pericyte surrounding vascular endothelium, the tightness of theconnection between endothelial vessels and the size of the gap, thepresence of fenestrae in the wall of endothelial vessels, and thepresence of diaphragm in the fenestrae, etc., all affect the vascularbarrier structure and permeability; more further control the efficiencyand degree of substance exchange and cell migration in blood-tissue. Theendothelium surrounded by pericyte, connected tightly, and withoutfenestrae is the least permeable, and the efficiency of substanceexchange and the degree of cell migration are also the lowest; theendothelium without pericyte, not completely continuous, and withfenestrae in the wall (such as hepatic sinuses area) have the highestpermeability, and thus the efficiency of substance exchange and thedegree of cell migration are the highest; the permeability of porousendothelial vessels containing diaphragm is generally lower than that ofporous endothelial vessels without diaphragm.

The only substance known to constitute the fenestral diaphragm orstomatal diaphragm in endothelial vessels until now is plasmalemmavesicle-associated protein (PLVAP). PLVAP simply PV-1, is a glycoproteinspecifically expressed in endothelial vessels; its cDNA and the aminoacid sequences coding protein were first cloned from rat lung tissue byStan R V et al. and reported (Stan R V, Ghitescu L, Jacobson B S, PaladeG E: 1999 J Cell Biol. 145:1189-9 ; Stan R V, Kubitza M, Palade G E.1999 Proc Natl Acad. Sci 96:13203-43207). After that, Stan R V et al.reported PLVAP/PV-1 gene, its cDNA and amino acid sequences codingprotein in human and mouse again (Stan R V, Arden K C, Palade G E 2001Genomics 72: 304-313 ; Review Stan RV 2007 Endothelial stomatal andfenestral diaphragms in normal vessels and angiogenesis. J Cell Mol Med.621-643).

PV-1 is a single transmembrane transmembrane protein; the molecularweight is around 55-60 kD. PV-1 protein in rat and mouse has afull-length of 438 amino acids (human PV-1 protein has 442 amino acids),and its intracellular region is relatively short (including 27 aminoacids), located at the N-terminal. The C-terminal extracellular regionis longer (including 358 amino acids) and exposed to the vascular lumen.

In a normal physiological state, except highly expressed in someendocrine glands, such as pituitary gland, adrenal, Choroid plexus ofbrain or retina, and lung tissue, PV-1 are low expressed in othertissues of the body (generally only maintain background expression) orno expression (Hnasko R et al., 2002 J Endocrinol 1.75:649-61). However,PV-1 expression is significantly upregulated in tumor tissues,hypoxia/trauma, and inflammation accompanied by angiogenesis.

Vascular endothelial growth factor (VEGF) (Leung D W et al., 1989Science 246:1306-09, also named as vascular permeability factor, (VPF)(Keck P J et al., 1989 Science 246:1309-12) is known as the strongestand the most important substance stimulating angiogenesis and osmosisuntil now (Carmeliet P et al., 1996 Nature 380: 435-438; Ferrara N etal., 19% Nature 380: 439-412). VEGF/VPF is also known as the mostimportant factor inducing the formation of vascular cortical microporousstructure and upregulating the expression of PV-1 gene (Roberts W G andPalade G E. 1995 J Cell Sci. 108:2369-2379: Roberts W G and Palade G E.1997 Cancer Res. 57:765-772 ; Roberts W G et al. 1998 Am J Pathol.153:1239-48 ; Esser S et al., 1998 J Cell Biol 140:947-959, Strickland LA et al., 2005 J Pathol. 206:466-475 ; Ioannidou S et al. 2006 Proc NatlAcad Sci 103: 16770-16775).

Other factors, such as tumor necrosis factor-α. (TNF-α), interleukin-6(IL-6), and oncogenic factor phorbol myristate acetate (PMA), etc., alsostimulate the formation of vascular cortical microporous structure andupregulate the expression of PV-1 gene (Lombardi T et. al. 1986 JCB102:1965-4970; Stan R V et al. 2004 Mol Biol Cell 15: 3615.-3630 ;Strickland L A et al., 2005 J Pathol. 206:466-475).

The earliest study report regarding PV-1 physiological functionoriginated from an article by Keuschnigg J et al. published in Blood in2009 (Keuschnigg J et al., Blood. 114:478-84. The prototype endothelialmarker PAL-E is a leukocyte trafficking molecule). PAL-E is a code nameof a murine monoclonal antibody, and its full name is PathologischeAnatomic Leiden-endothelium (Schlingemann R O et al., 1985 Lab Invest.52:71-6), the antigens it recognizes are mainly specific to bloodvessels; Niemela H et al. reported the antigen recognized by PAL-Emonoclonal antibody is human plasmalemma vesicle-associated protein(PV-1) (Niemela H et al., 2005 Blood.;106:3405-3409). Keuschnigg J etal. discovered that in human umbilical vein endothelial cells (HUVEC)activated by TNF-α, PAL-E/PV-1 protein significantly gathers around theendothelial cell membrane and surrounds the lymphocytes crossing theumbilical vein endothelial cells; PAL-E/PV-1 antibody was added toinhibit the transmigration of lymphocytes. In a mouse model of acuteperitonitis and balloon inflammation, the number of mononuclear orlymphocyte cells in the abdominal cavity of mice was reduced by up to85% after the injection of an antibody with a code-name MECA-32 throughthe tail vein (Keuschnigg J et al,, 2009 Blood. 114:478-84).

The importance of PLVAP/PV-1 in the formation of the diaphragm inendothelial vascular micropores and regulating vascularbarrier/permeability have recently clearly demonstrated in knockoutmice. As reported by Stan R V et al. in Dev Cell in 2012, in PV -1knockout mice, embryos could not survive under C57BL/6N. In a mixedgenetic background, a few embryos survive to birth. PV-1 gene knockoutmouse was unable to form intravascular cortical microporous membrane orconcave membrane. The absence of the diaphragm increases the leakage ofendothelial cells, results to a large amount of protein leaking outsideof the blood vessels, tissue edema, and death of the born animal inearly development due to severe non-inflammatory protein-loss enteritis(Stan R V et al., 2012 Dev Cell. 23:1203-18)

Similarly, Herrnberger L et al. reported in Histochem Cell Biol in 2012that Plvap (PV-1) gene knockout mouse homozygous (Plvap /−) embryos withC57BL/6N genetic background died before birth, with abnormalities, suchas subcutaneous edema, hemorrhage, and defective subcutaneous capillarywalls. Also, Plvap−/−embryonic hearts showed ventricular septal defectsand thinner ventricular walls. In the C57BL/6N/FVB-N mixed geneticbackground, Plvap embryos can develop to birth, but the mouse born canonly live for a maximum of 4 weeks (Herrnberger L et al., 2012 HistochemCell Biol. 138:709-24).

Under normal conditions, the area in the body existing vascular-tissuebarriers, such as blood-brain barrier in the central nervous system, andblood-retinal barrier in eyes, there is no Plasma membrane pores on thewall of the endothelial vessel and no expression of PV-1/PAL-E antigen.However, under some pathological state, such as ischemic stroke, spinalcord injury, experimental allergic encephalomyelitis (EAE)/multiplesclerosis (MS), primary or metastatic brain tumors, diabeticretinopathy, etc. the structure of vascular-tissue barriers in theseareas are often destroyed, and there are micropores in the wall of theendothelial vessels accompany with the upregulation of PV-1/PAL-E(Carson-Walter E B et al., 2005, Clin Cancer Res. 11:7643-50; Shue E Het al., 2008 BMC Neurosci 9:29; Mozer A B et al., 2010 Curr NeurovascRes. 7:238-508). For instance, Shue E H et al. found that in acutecerebral ischemia model induced by cerebral artery embolization inmouse, PV-1/PAL-E antigen began to express in a small number of cerebralvessels in the embolized area after 48 hours of acute cerebral ischemiaoccurring; on the 7th day, the expression of PV-1/PAL-E antigen in theembolized area reached its peak (Shue E H et al. 2008 BMC Neurosci9:29).

Similarly, Schlingemann R O et al. found that in patients of diabeticretinopathy and diabetic mice Akimba with damaged vascular-retinabarrier structure, there is PV-1/PAL-E antigen upregulated expression inthe retinal endothelial vascular wall, and the level of upregulatedexpression is positively correlated with the degree of damage andpermeability of the vascular-retinal barrier structure (Schlingemann R Oet al., 1999, Diabetologia. 42:596-602 ; Wisniewska-Kruk J et al., 2014,Exp Eye Res. 122:123-31). Inhibiting the expression of PV-1 gene inendothelial cells through lentivirus-mediated silencing of interferingRNA (siRNA) techniques can prevent or reduce the formation ofVEGF-induced endothelial vascular membrane micropores/caveolae anddamage to the structure of vascular-retinal barrier (Wisniewska-Kruk Jet al. 2016 Am j Pathol, 186:1044-54)

Therefore, PLVAP (PV-1) is not only the main component formingendothelial vascular fenestral diaphragm and stomatal diaphragm, butalso support endothelial vascular fenestrae or caveolae structure, butalso directly participate in regulating angiogenesis and osmosis.

SUMMARY

A technical problem to be solved in the present invention is to providean antibody or a derivative thereof specifically recognizing and bindinghuman plasmalemma vesicle-associated protein (PLVAP, or PV-1 for short),such as the Fab fragment of an antibody, an Fv fragment, a single-chainantibody, a bi-specific antibody, an antibody-drug conjugate (ADC), andchimeric antigen receptor T-cell (CAR-T), etc.

The antibody or derivative thereof can be used as a main activecomponent alone and prepared into an appropriate pharmaceuticalformulation to interfere with angiogenesis/osmosis mediated by PLVAP(PV-1), to reach the effects of curing or delaying the occurrence anddevelopment of related diseases. The diseases closely related toangiogenesis/osmosis are suitable for treating with the antibody,including various malignant tumors, age-related macular degeneration(AMD), or diabetic retinopathy such as diabetic macular edema (DME),etc.

Anti-PLVAP (PV-1) antibody can also be used sequentially or incombination with other drugs currently on the market or underdevelopment when treating the above disease.

A second technical problem to be solved in the present invention is toprovide a DNA molecule or gene coding the above antibody.

A third technical problem to be solved in the present invention is toprovide a pharmaceutical compound or a pharmaceutical compositioncomprising the above antibody.

A fourth technical problem to be solved in the present invention is toprovide a use of the pharmaceutical compound or the pharmaceuticalcomposition for the treatment of angiogenesis or osmosis-relateddiseases especially choroidal neovascularization fundus diseases.

A sixth technical problem to be solved in the present invention is toprovide a reagent or a kit comprising the above antibody for detectingand analyzing PLVAP (PV-1) protein or tracking and labeling the tissuecells expressing PLVAP (PV-1) positively in vivo or in vitro.

A seventh technical problem to be solved in the present invention is toprovide a preparation method of the above antibody.

PLVAP (PV-1) antigen, in general, is only selectively expressed in thefenestrae of the vascular wall in the lesion area under pathologicalconditions such as inflammation, tumor, and diabetic retinopathy, etc.Therefore, if the antibody specifically recognizing PLVAP (PV-1) proteinis given into the body, the antibody can cross-link or combine with thediaphragm of the vascular wall fenestrae to form a physical blockage orclosure of the fenestrae of the vascular wall, thus preventing orreducing vessel penetration/leakage. The antibody or derivative thereof,specifically recognizing and binding PLVAP (PV-1) protein on the wall ofvascular endothelium as an active component, can be prepared into anappropriate pharmaceutical formulation to treat or interfere withangiogenesis/osmosis-related diseases. These antibodies or derivativesthereof can also be used as a targeting carrier due to specificallygathering and binding to the walls of new vessels or endothelialvessels. The antibodies or derivatives thereof conjugate or wrap withother drugs, such as anti-tumor chemical drugs, radioactive drugs, ortoxin, to form antibody-drug conjugate (ADC), and transported andgathered together in the lumen of new vessels in the tumor area andachieved dual effects of blocking the vessels in tumor area and killingtumor cells with drugs. The antibody or derivative thereof specificallybinding PLVAP (PV-1) antigen, such as antibody-drug conjugate (ADC), canstill be used sequentially or combined with other drugs on the market orunder development to treat angiogenesis/osmosis-related diseases.

To resolve the above technical problems, the present invention adoptsthe following technical solutions:

In one aspect, the present invention provides a brand-new monoclonalantibody or a derivative thereof, specifically binding human plasmalemmavesicle-associated protein extracellular membrane area The monoclonalantibody or the derivative thereof comprises a first variable region anda second variable region, wherein the first variable region is anantibody light chain variable region comprising antigencomplementarity-determining regions CDR1, CDR2 and CDR3 having aminoacid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ IDNO: 19, respectively; and wherein the second variable region is anantibody heavy chain variable region comprising antigencomplementarity-determining regions CDR1, CDR2 and CDR3 having aminoacid sequences as set forth in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ IDNO: 24, respectively.

The monoclonal antibodies include a murine antibody, a human-mousechimeric antibody, and a humanized antibody, etc.; the derivativesinclude a Fab fragment of an antibody, an Fv fragment, a single-chainantibody, a bi-specific antibody, an antibody-drug conjugate, andchimeric antigen receptor T-cell (CAR-T), etc.

As a preferred technical solution, the first variable region is anantibody light chain variable region having an amino acid sequence asset forth in SEQ ID NO: 16; and the second variable region is anantibody heavy chain variable region having an amino acid sequence asset forth in SEQ ID NO: 21.

As a preferred technical solution, the antibody or derivative thereofcomprises the antibody light chain variable region, a human antibodylight chain constant region, the antibody heavy chain variable region,and a hinge region of a human antibody heavy chain constant region, CH1region, CH2 region, and CH3 region.

As a preferred technical solution, the human antibody light chainconstant region is a kappa chain or a lambda chain of a human antibody;the human antibody heavy chain constant region is a human IgG1 isotype,IgG2 isotype, IgG3 isotype, IgG4 isotype, IgA, or IgM, wherein IgG1isotype or IgG4 isotype are more preferred.

In a second aspect, the present invention provides a nucleotide sequencecoding a DNA molecule or gene of the antibody or derivative thereof, thenucleotide sequence of the antibody light chain variable region is setforth in SEQ ID NO: 15, the nucleotide sequence of the antibody heavychain variable region is set forth in SEQ ID NO: 20.

In a third aspect, the present invention provides an expression vectorcomprising a nucleotide sequence coding the DNA molecular/gene of theabove antibody or derivative thereof and an expression regulatorysequence operably linked to the sequence.

In a fourth aspect, the present invention provides a recombinant hostcell transfected with the above expression vector. The recombinant hostcell or a progeny cell thereof expresses the above antibody orderivative thereof. The antibodies include a murine antibody, ahuman-mouse chimeric antibody, and a humanized antibody, etc.; thederivatives include a Fab fragment of an antibody, an Fv fragment, asingle-chain antibody, a bi-specific antibody, an antibody-drugconjugate (ADC), or chimeric antigen receptor T-cell (CAR-T).

In a fifth aspect, the present invention provides a pharmaceuticalcompound or a pharmaceutical composition comprising a pharmaceuticallyeffective amount of the antibody or derivative thereof and apharmaceutically accepted carrier or recipient.

In a sixth aspect, the present invention provides a use of the abovepharmaceutical compound or the pharmaceutical composition for thepreparation of a medicament for the treatment of angiogenesis orosmosis-related diseases. The angiogenesis or osmosis-related diseasesinclude various malignant tumor and choroidal neovascularization fundusdisease, such as age-related macular degeneration (ADM), diabeticretinopathy such as diabetic macular edema (DME) and retinal veinocclusion, etc.

As a preferred technical solution, the pharmaceutical composition alsocomprises a pharmaceutically effective amount of active componentantagonizing and blocking vascular endothelial growth factor (VEGF) orits receptor (VEGF-R) and a pharmaceutically accepted carrier. PLVAP(PV-1) antibody in the present invention as a pharmaceutical preparationcomponent in treating angiogenesis or osmosis-related diseases such asvarious malignant tumor and choroidal neovascularization fundus disease,can also be used sequentially or combined with drugs targeting VEGFand/or VEGF-R The preferred targeting VEGF and/or VEGF-R drugs includemacromolecular biological drugs such as anti-VEGF monoclonal antibodyBevacizumab (brand name: Avastin), anti-VEGF monoclonal antibody Fabfragment Ranibizumab (brand name: Lucentis), anti-VEGFR2 monoclonalantibody Ramucirumab (brand name: Gyramza) and anti-hVEGF monoclonalantibody hPV19 (under development in Suzhou Stainwei Biotech Inc., seeChinese patent document: ZL 201210540692X, patent name: monoclonalantibody for antagonizing and inhibiting the binding of vascularendothelial growth factor to its receptor, as well as coding sequenceand use; American granted patent document: U.S. Pat. No. 9,580,498 B2),VEGFR-Fc fusion protein drug such as Albercept (brand name: Eylea) andConbercept, etc.; the preferred small molecular chemical drugs includeSunitinib, Sorafenib, Apatinib, and Pazopanib, etc.

As a preferred technical solution, the PLVAP (PV-1) antibody in thepresent invention is used for fundus disease as a local administration,mainly depending on specific binding of the antibody and the diaphragmof the vascular wall fenestrae to form a physical blockage or closure ofthe vascular wall fenestrae, thus preventing or reducing vesselpenetration. Therefore, as a pharmaceutical component, the antibody canbe more consideration on preparation wild-type or the constant region ofhuman genetically modified IgG4 or IgG2 isotype antibody, or an antibodyFab-fragment, an Fv fragment, or a single-chain antibody withoutconstant region, etc., to reduce or eliminate antibody-dependentcellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC),further reduce the direct killing of blood vessels or tissue cells inthe treatment area. The wild type or the constant region of humangenetically modified IgG4 or IgG2 isotype antibody, or antibody aFab-fragment, Fv fragment, or a single-chain antibody without constantregion, etc. can be cloned or synthesized in vitro respectively bygenetic engineering technology known to the skilled in the art.

As another preferred technical solution, the PLVAP (PV-1) antibody inthe present invention is used for tumor; the antibody can be moreconsideration on preparation wild-type or the constant region of humangenetically modified IgG1 or IgM isotype antibody to maintain orincrease ADCC or CDC of antibody, further achieve a stronger effect ofkilling tumor tissue and cells. The wild-type or the constant region ofhuman genetically modified IgG1 or IgM isotype antibody can be cloned orsynthesized in vitro by genetic engineering technology known to theskilled in the art.

PLVAP (PV-1) antibody or a derivative thereof in the present inventioncan be used as a targeting carrier due to specific binding to the newendothelial vessels or the walls of vessels in the tumor area. Theantibody or derivative thereof conjugates or wraps with other anti-tumordrugs or toxins to form antibody-drug conjugate (ADC), then transportedand gathered together in the lumen of new vessels in the tumor area andachieved better killing tumor effects. The conjugating or wrappingmethod of antibody and drugs or toxins can take the conventionaltechniques known to people in this field. This antibody-drug conjugateespecially suits some areas the common medicines can not reach, such asbrain tumors, including primary brain tumors such as glioblastoma ormetastatic brain tumors. PLVAP (PV-1) antibody or antibody-drugconjugate can be combined with oral small molecular drugs such astemozolomide when used for brain tumors such as glioblastoma. PLVAP(PV-1) antibody or antibody-drug conjugate in the present invention isalso particularly suitable for some malignant tumors with relativelyhigh PLVAP/PV-1 gene expression, such as primary liver cancer andmetastatic liver cancer. This antibody-drug can also be administered bylocal injection into blood vessels in the liver, achieving more accuratetargeted therapies and reducing side effects in other parts of the body.

As another preferred technical solution, the PLVAP (PV-1) antibody inthe present invention can also be used sequentially or combined withmonoclonal antibody drugs targeting inhibitory immune checkpointmolecules for various malignant tumors, including primary (e.g.,glioblastoma) or metastatic brain tumor, lung cancer, gastric/esophagealcancer, liver cancer, kidney cancer, cervical cancer, etc. The preferredmonoclonal antibody drugs targeting inhibitory immune checkpointmolecules used sequentially or in combination with PLVAP (PV-1) antibodyinclude anti-CTLA4 (Cytotoxic T-lymphocyte Antigen-4) antibody,Ipilimumab (brand name: Yervoy), anti-PD-1 (programmed death protein 1)antibody, Nivolumab (brand name: Opdivo), Pembrolizumab (brand name:Keytruda), and the monoclonal antibody code-named hAB21. (underdevelopment in Suzhou Stainwei Biotech Inc. See PCT patent, applicationdocument: PCT/CN2017/089282, monoclonal antibody antagonizing andinhibiting binding between human PD-1 antigen and ligand thereof,preparation method thereof and application thereof), anti-PD-L1monoclonal antibody drugs include Atezolizumab (brand name: Tecentriq),Avelumab (brand name: Bavencio) , Durvalumab (brand name: imfinzi), etc.

As another preferred technical solution, the PLVAP (PV-1) antibody inthe present invention can be firstly prepared into chimeric antigenreceptor T-cell (CAR-T), then introduced into the immune cells isolatedfrom peripheral blood of tumor patients, such as T-lymphocytes. Afterculturing and amplification in vitro, these lymphocytes recognizingPLVAP (PV-1) antigen were injected back into the body to achieve theeffect of treating the tumor by targeting the vascular endothelial cellsand new blood vessels in the tumor area. Comparing with normal CAR-Tdirectly targeting tumor antigen such as CD19 or CD20, CAR-T in thepresent invention, specifically targeting the vascular endothelial cellsand new blood vessels in the tumor area, does not rely on the expressionof tumor antigen, can be used for several types of solid tumors. Thepreparation of PLVAP (PV-1) antibody in the present invention intochimeric antigen receptor T-cell (CAR-T) can take conventionaltechniques s known to a person skilled in the art.

In a specific example of the present invention, the use of human-mousechimeric PLVAP (PV-1) antibody as a single component or combination withanti-VEGF antibody in the treatment of choroidal neovascularizationfundus diseases in Macaca Fascicularis is depicted.

In a seventh aspect, the present invention provides a monoclonalantibody or a derivative thereof binding both human and monkeyplasmalemma vesicle-associated protein, wherein the antibody bindsantigens having amino acid sequences as set forth in SEQ ID NO: 8 or SEQID NO: 25, and competitively binds PV-1 with the antibody or derivativethereof.

In an eighth aspect, the present invention provides a method ofantagonizing and blocking angiogenesis or osmosis in vivo mediated byplasmalemma vesicle-associated protein, which is administering anappropriate amount of the antibody or derivative thereof.

In a ninth aspect, the present invention provides a detecting reagent ora detecting kit comprising the antibody or derivative thereof fordetecting and analyzing plasmalemma vesicle-associated protein in tissueor cell sample or tracking the tissue cells expressing PLVAP (PV 1)positively in vivo or in vitro.

In a tenth aspect, the present invention provides a method for preparingthe above antibody or derivative thereof, and the method comprises thefollowing steps:

e) Providing an expression vector comprising the DNA sequence coding theantibody or its derivative and an expression regulatory sequenceoperably linked to the DNA sequence;

f) Transfecting a host cell such as CHO cell with the expression vectorof step a);

g) Culturing the host cell from step b) under conditions suitable forthe expression of the antibody; and

h) Isolating, purifying, and collevting the antibody from a host cellculture medium by affinity chromatography.

The term “monoclonal antibody (mAb)” used herein refers to animmunoglobin obtained from a clonal cell, with the same structure andchemical characteristics and specific to a single antigenic determinant.The monoclonal antibody is different from a regular polyclonal antibodypreparation (usually having different antibodies directed againstdifferent determinants). Each monoclonal antibody is directed against asingle determinant of an antigen. In addition to its specificity, themonoclonal antibody is also advantageous because it is cultured fromhybridoma or recombinant engineering cells and will not be mixed withother immunoglobulins. The modifier “monoclonal” indicates that theantibody's properties are achieved from a homogeneous population ofantibodies, which should not be interpreted as any special method thatneeds to be used for production of antibodies.

The term “humanized monoclonal antibody” as used herein refers to thatall or most of the amino acid sequences of the murine monoclonalantibodies (including the framework region sequence in the variableregion), except complementarity-determining regions (CDR) aresubstituted by the amino acid sequences of human immunoglobulins, toreduce the immunogenicity of the murine monoclonal antibody to theutmost extent by genetic engineering methods.

The terms “antibody” and “immunoglobulin” used herein refer to aniso-tetra proteoglycan of about 150,000 Daltons with the same structuralcharacteristics and consist of two identical light chains and twoidentical heavy chains. Each light chain is linked to the heavy chainthrough a covalent disulfide bond, while the same isotype heavy chainsof the different immunoglobulins have a different amount of disulfidebonds. Each heavy chain and each light chain also have regularly spacedintrachain disulfide bonds. Each heavy chain has a variable region(V_(H)) at one end, followed by several constant regions. Each lightchain has a variable region (V_(L)) at one end, and a constant region atthe other end. The constant region of the light chain is opposite to thefirst constant region of the heavy chain. The variable region of thelight chain is opposite to the variable region of the heavy chain.Special amino acid residues form an interface between the variableregion of the light chain and the heavy chain.

The term “variable” used herein indicates that some portion of thevariable region in an antibody are different in sequence, which resultsin binding and specificity of various specific antibodies to thespecific antigens. However, variability is not evenly distributedthroughout the whole antibody variable region. Instead, it concentrateson three fragments in the complementarity-determining region (CDR) andhypervariable region in the light-chain or heavy-chain variable regions.The more conservative part of the variable region is called theframework regions (FR), There are four FR regions in each variableregion of the heavy-chain and light-chain of an antibody. The FR regionsare roughly in a β-folded configuration and connected by three CDRsforming a connecting loop. The partial β-folded configuration can formin some cases. The CDRs in each chain are close together through the FRregions and form the antigen-binding site of the antibody together withthe CDRs of another chain (see Kabat et al, NIH Publ. No. 91-3242, Vol.1, pp. 647-669 (1991)). The antibody's constant region does not directlyparticipate in the binding of the antibody to the antigen. Still, itexhibits different effects and functions, such as participating inantibody-dependent cytotoxicity (ADCC) and complement mediatedcytotoxicity (CDC) of the antibody.

The antibody of the present invention can be usually prepared by thefollowing methods:

Firstly, insert the gene coding the antibody in the present inventioninto the expression vector containing a suitable expression regulatorysequence.

The term “expression regulatory sequence” used herein usually refers toa sequence that participates in the control of the gene expression. Theexpression regulatory sequence includes a promoter operable linked tothe target gene and a termination signal. The gene (DNA) sequence of thepresent invention's antibody in can be encoded by the common techniqueswell known by the skilled in the art, such as artificial synthesisaccording to the protein sequences disclosed by the present invention orthe PCR amplification. After that, the DNA fragments synthesized oramplified by the PCR method can be inserted into a suitable expressionvector by various methods well known in the art. The expression vectorused in the present invention can be available on the market and wellknown for those skilled in the art, such as the pCDNA3.1 expressionvector from Invitrogen.

The suitable host cells for accepting the expression vectortransformation generally include both prokaryotes and eukaryotes.Commonly used prokaryotes host cells include E. coli, and Bacillussubtillis, etc. Commonly used eukaryotes host cells include yeast cells,insect cells, and mammalian cells. In the present invention, thepreferred host cells are mammalian, particularly Chinese hamster ovary(CHO) cells.

The host cells transfected by the expression vector are cultured undersuitable conditions (e.g., culturing with a serum-free culture medium ina cell culture flask or bioreactor by adhesion to the wall orsuspension). The supernatant is collected and purified by commonseparation steps or means well known by the skilled in the art,including protein-A affinity chromatography, ion-exchangechromatography, filtration, etc. to produce the antibodies of thepresent invention.

The purified antibodies of the present invention can be dissolved in anappropriate solvent such as sterile saline liquid. The solubility can beprepared between 0.01 and 100 mg/mL. The ideal final solubility can beprepared between 1 mg/ml and 40 mg/ml.

To obtain a murine monoclonal antibody specifically binding PLVAP (PV-1)protein as well as the hybridoma cell line secreting this antibody, thepresent invention chose recombinant human PV-1 protein extracellularmembrane area expressed by the mammalian cell (CHO) as an immune antigenand immunized mice to obtain the anti-hPV-1 protein polyclonal antibodyby repeated small dose subcutaneous injection. The mice with high titersof antibody were selected to get the spleen cells, fused with a mousemyeloma cell line in vivo. After drug screening and subcloning, severalhybridoma monoclonal cells secreting the antibody of anti-human PV-1protein were established. A mouse hybridoma clone coded STW-139-15 wastested by ELISA, immunohistochemistry, flow Cytometer, and other manymethods and proved that the monoclonal antibody secreted by thisantibody could specifically bind PV-1 protein not only in normal humantissues and tumor tissues but also in monkey tissues.

The gene sequences coding the heavy-chain and light-chain variableregion protein of murine antibody were cloned from the mouse hybridomaSTW-139-15 cell line by genetic engineering methods, etc. in the presentinvention. The present invention completed the humanization of theantibody on the above basis to obtain human-mouse chimeric antibodySTW-139-15-C and the expression vector. The expression vector wastransfected into Chinese hamster ovary (CHO) cells to obtain therecombinant engineering cells secreting the human-mouse chimericantibody stably and efficiently. The recombinant engineering cells werecultured on a large scale, and the culture supernatant was harvested.After centrifugation and filtration with a 0.45 μm filtration membrane,the supernatant was isolated and purified by Protein-A affinitychromatography, and the purified human-mouse chimeric antibodySTW-139-15-C protein was obtained.

The purified antibody STW-139-15-C protein was filtrated and eliminatedbacteria, dissolved in appropriate solvent again, and prepared intopharmaceutical preparations, which can be used in vivo and in vitro totest its biological or pharmacological activities.

One method of testing pharmacological activities of human-mouse chimericantibody in vivo is to use Macaca Fascicularis choroidneovascularization disease model induced by laser irradiation,administered through vitreous injection. Examine the inhibition effectof ST1,17-139-15-C antibody administered alone or combined withanti-VEGF antibody-drug on choroidal neovascularization leakage andgrowth. Compare with the inhibition effects of anti-VEGF antibodyadministered alone. The test results showed that STW-139-15C monoclonalantibody specifically binding PLVAP/PV-1, no matter administered aloneor in combination with anti-VEGF antibodies, had a significantinhibitory effect on laser-induced chorionic neovascularization inMacaca Fascicularis and could be used to treat diseases related toangiogenesis/osmosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the amino acid sequence comparisonanalysis of human PV-1 protein and mouse PD-1 protein in Example 1 ofthe present invention.

FIG. 2 is the SDS-PAGE electrophoretic analysis of the human PV-1-Hisrecombinant protein; lane 1 is a DTT-reduced sample; the markerrepresents the protein molecular weight standard substance (kd).

FIG. 3A is a schematic diagram of the representative results ofdetermining the supernatant sample of unfused SP2/0 myeloma cell(negative control) specifically binding to CHO cells (CHO/PV-1)transiently transfected with human PV-1 gene by Immunohistochemistry(IHC) method in Example 1 of the present invention. FIG. 3B is aschematic diagram of the representative results of determining the serumof the human immunized with PV-1 antigen (diluted at 1:200) specificallybinding to CHO cells (CHO/PV-1) transiently transfected with human PV 1.gene by Immunohistochemistry (IHC) method in Example 1 of the presentinvention. FIG. 3C is a schematic diagram of the representative resultsof determining the cell culture supernatant sample of the mousehybridoma STW-139-15 specifically binding to CHO cells (CHO/PV-1)transiently transfected with human PV-1 gene by Immunohistochemistry(IHC) method in Example 1 of the present invention.

FIG. 4 is a schematic diagram of the results of the ELISA in Example 2of the present invention, which shows the binding of the supernatantsample of the mouse hybridoma cell STW-139-15 and the recombinant humanPV-1 extracellular membrane protein coated in a 96-well plate. MAb113 isa non-related mouse monoclonal antibody sample (anti-SOST antibody); thenegative control is culture supernatant sample of unfused. SP2/0 myelomacell.

FIG. 5 is a schematic diagram of the comparison and analysis results bythe ELISA method in Example 3 of the present invention, which shows thebinding of the mouse monoclonal antibody sample STW-139-15 and therecombinant human PV-1-Fc fusion protein and recombinant proteins ofseveral other non-related genes.

FIGS. 6A-6D are schematic diagrams of the representative results testedby the flow cytometer in Example 4 of the present invention, whichdetermines the binding of the mouse monoclonal antibody STW-139-15sample and the CHO cells steadily transfected with human PV-1 geneCHO/PV-1). FIG. 6A is a schematic diagram of the representative resultsdetermining the binding of the culture supernatant sample of unfusedSP2/0 myeloma cell (as a negative control); FIG. 6B is a schematicdiagram of the representative results determining the binding of anon-related mouse monoclonal antibody sample mAb21 (anti-PD-1 Mab) andthe CHO cells steadily transfected with human PV-1 gene CHO/PV-1). FIG.6C is a schematic diagram of the representative results determining thebinding of the serum of mouse immunized with human PV-1 protein (dilutedat 1:200 as a positive control) and the CHO cells steadily transfectedwith human PV-1 gene CHO/PV-1). FIG. 6D is a schematic diagram of therepresentative results determining the binding of the cell culturesupernatant sample of the mouse hybridoma STW-139-15 (the mousemonoclonal antibody STW-139-15 sample) and the CHO cells steadilytransfected with human PV-1 gene CHO/PV-1).

FIG. 7 is a dose-response curve of antibody's solubility-meanfluorescence value tested by the flow cytometer in Example 4 of thepresent invention, which determines the binding of a series of gradientdilutions of murine STW-139-15 monoclonal antibody sample and CHO cellsteadily transfected with human PV-1 gene (CHO/PV-1).

FIGS. 8A-8C are schematic diagrams of the representative results testedby the flow cytometer in Example 4 of the present invention, whichdetermines and analyzes the binding of the mixture sample containingmurine STW-139-15 monoclonal antibody samples, CHO cells and CHO/PV-1cells (at a ratio of 9:1). FIG. 8A is a schematic diagram of therepresentative results determining the binding of the culturesupernatant sample of unfused SP2/0 myeloma cells (as a negativecontrol) in the mixture sample containing murine STW-139-15 monoclonalantibody samples, CHO cells and CHO/PV-1 cells (at a ratio of 9:1). FIG.8B is a schematic diagram of the representative results determining thebinding of a non-related mouse monoclonal antibody sample mAb21(anti-PD-1 Mab) in the mixture sample containing murine STW-139-15monoclonal antibody samples, CHO cells and CHO/PV-1 cells (at a ratio of9:1). FIG. 8C is a schematic diagram of the representative resultsdetermining the binding of the murine monoclonal antibody STW-139-15sample in the mixture sample containing murine STW-139-15 monoclonalantibody samples, CHO cells and CHO/PV-1 cells (at a ratio of 9:1).

FIG. 9 is a schematic diagram of the representative results tested bythe flow cytometer in Example 5 of the present invention, whichdetermines and analyzes the binding of the murine monoclonal antibodySTAN-139-15 sample and human HUVEC. A01NC is the culture supernatantsample of unfused SP2/0 myeloma cells (as a negative control).

FIGS. 10A-10F are schematic diagrams of the representative results byImmunohistochemistry (IHC) method in Example 6 of the present invention,which determines the binding of the murine monoclonal antibodySTW-139-15 sample and tissue sections of the normal tissue. FIG. 10Adepicts the lung tissue sections. FIG. 10B depicts the liver tissuesections. FIG. 10C depicts the brain tissue sections. FIG. 10D depictsthe pancreatic tissue sections. FIG. 10E depicts the heart tissuesections. FIG. 10F depicts the spleen tissue sections.

FIGS. 11A-11F are schematic diagrams of the representative results byImmunohistochemistry (IHC) method in Example 7 of the present invention,which determines the binding of the murine monoclonal antibodySTW-139-15 sample and tissue sections of the human tumor tissue. FIG.11A depicts the lung cancer tissue sections. FIG. 11E depicts the livercancer tissue sections; FIG. 11C depicts the brain tumor tissuesections. FIG. 11D depicts the pancreatic cancer tissue sections. FIG.11E depicts the ovarian cancer tissue sections. FIG. 11F depicts thelymphoma tissue sections.

FIG. 12 is a schematic diagram of amino acid sequences comparison andanalysis of human PV-1 protein and monkey PV-1 protein in Example 8 ofthe present invention.

FIGS. 13A-13C are schematic diagrams of the representative resultstested by the flow cytometer in Example 8 of the present invention,which determines the binding of the murine monoclonal antibodySTW-139-15 sample and the CHO cell steadily transfected with monkey PV-1gene (CHO/monkey PV-1). FIG. 13A is a schematic diagram of therepresentative results determining the binding of the culturesupernatant sample of unfused SP2/0 myeloma cell (as a negative control)and the CHO cell steadily transfected with monkey PV-1 gene (CHO/monkeyPV-1). FIG. 13B is a schematic diagram of the representative resultsdetermining the binding of a non-related mouse monoclonal antibodysample mAB7 (anti-PD-1 Mab) and the CHO cell steadily transfected withmonkey PV-1 gene (CHO/monkey PV-1). FIG. 13C is a schematic diagram ofthe representative results determining the binding of the murinemonoclonal antibody STW-139-15 sample and the CIO cell steadilytransfected with monkey PV-1 gene (CHO/monkey PV-1).

FIG. 14 is a schematic diagram of the results tested by the ELISA methodin Example 10 of the present invention, which determines the binding ofthe human-mouse chimeric antibody STW-139-15-C sample and therecombinant human PV-1 extracellular membrane protein coated in a96-well plate.

FIGS. 15A to 15D are fundus fluorescein images of Macaca Fascicularis byvitreous injection of on the third week after photocoagulation at thetime points during the observation period in Example 11 of the presentinvention. FIG. 15A shows the images of the negative control group (0.9%NaCl injection). FIG. 15B shows the images of STW-139-15C monoclonalantibody tested sample group. FIG. 15C shows the images of the positivecontrol drug hPV19 Mab(anti-VEGF Mab). FIG. 15D shows the images ofcombination administration of TW-139-15C as a tested drug and positivecontrol drug hPV19 monoclonal antibody.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention will be further described in combination with theexamples. The following examples are offered by way of illustration onlyand are not intended to limit the invention.

EXAMPLE 1 Establishment and Screening Identification of Mouse HybridomaCell Line Secreting Anti-Human PV-1 Antibody

1.1 Amino Acid Sequence Comparison Analysis of Human PV-1 Protein andMouse PD 1 Protein.

The comparison analysis of the amino acid sequence of human PV-1 protein(NCBI Reference Sequence: NP_112600.1) (SEQ ID NO: 1)and the amino acidsequence of mouse PV-1 protein (NCBI Reference Sequence: NP_115774.2)(SEQ ID NO: 2) is shown in FIG. 1. More than 20 amino acids in theN-terminal located in the cell membrane (the sequence is marked initalics), the amino acid sequence of the transmembrane region of PV-1protein is marked in box and bold. The amino acid sequence of C-terminal(human: AA53-442; mouse: AA53-438) all located outside of cell membrane,wherein including 4 N-Glycisylation sites (marked in box) and 9Cysteines (marked in underline). There is only 62% homology in aminoacid sequences between human PV-1 protein and mouse PV-1 protein; thereare more than 100 amino acid difference sites in the extracellularregion. Therefore, it is speculated that the mouse antibody targetingthe human PV-1 extracellular antigen region can be prepared byimmunizing mice with the traditional antigen protein and hybridomapreparation techniques.

1.2 Expression and Preparation of the Recombinant Human PV-1 Protein(Immunogen)

In the example of the present invention, firstly collect the total RNAfrom human umbilical vein endothelial cells (HUVEC) and obtain cDNA byreverse transcription-polymerase chain reaction (RT-PCR). After that,the gene fragment coding human PV-1 protein was cloned by PCR technologyusing cDNA as the template. After DNA sequencing and identification,treated with restriction DNA endonuclease, cloned into DNA plasmid toexpress exogenous genes in CHO cells effectively, then the recombinantplasmid was obtained.

1.2.1 Cloning of the Gene Coding Human PV-1 Full-Length Protein andConstruction of Expression Plasmid Thereof

The construction process of the expression plasmid is as follows:

Firstly, the gene fragment coding human PV-1 full-length protein (about1344 bp in length) was successfully amplified by PCR using the abovecDNA as a template and the following pair of primers:

Forward primer hPV-1-His-F-HindIII: (SEQ ID NO: 3)AACTAAGCTTGCCACCATGGGTCTGGCCATGGAGCACGGA;Reverse primers hPV-1-His-R-XhoI: (SEQ ID NO: 4)ACCACTCGAGTCAGTGATGGTGATGGTGATGGCCACTGGATGGGGCTACA GGGAT

The DNA amplified by PCR was recycled and treated with the restrictionDNA endonuclease, cloned into the expression plasmid pCDNA3.1(Invitrogen), then the recombinant plasmid was obtained. After DNAsequencing and identification, treated with restriction DNAendonuclease, the recombinant plasmid effectively expressing exogenoushuman PV-1 genes in CHO cell membrane (Plasmid name: pQY-PV-1) wasobtained.

1.2.2 Construction of Expression Plasmid of the Human PV-1 ExtracellularMembrane Recombinant Protein with His-6 Label in C-Terminal

The gene fragment of the human PV-1 extracellular membrane protein with6 histidines label in C-terminal (PV-1-His) was successfully amplifiedby using PCR recycled product in the previous section (1.2.1) as atemplate and the following pair of primers:

Forward primer hPV-1-Fc-F-BglII: (SEQ ID NO: 5)GTGGAGATCTCACGTGAGCACAGAGTCCAACCTG; Reverse primer hPV-1-His-R-XhoI:(SEQ ID NO: 4)

GGGAT

The DNA amplified by PCR was recycled and treated with the restrictionDNA endonuclease, transferred into the expression vector pCDNA3.1-DHFRwith a signal peptide, then the recombinant plasmid was obtained. Therecombinant plasmid secreting the recombinant gene hPV-1-His in CHOcells (name: pQY-DHFR-PV1-His) was successfully obtained by endonucleasedigestion and DNA sequencing identification.

1.2.3 Construction of the Recombinant Human PV-1-Fc Fusion ProteinExpression Plasmid

The construction process of the expression plasmid was as follows:

The gene fragment of hPV-1 extracellular membrane region (about 1176 byin length) was successfully amplified by PCR using PCR recycled productin the previous section (1.2.1) as a template and the following pair ofprimers:

Forward primer hPV-1-Fc-F-BglII: (SEQ ID NO: 5)GTGGAGATCTCACGTGAGCACAGAGTCCAACCTG Reverse primer hPV-1-Fc-R-BamHI:(SEQ ID NO.: 6) GTGGGCATGTGTGAGTGGATCCGCCACTGGATGGGGCTACAG

After that, the recombinant gene (about 1859 bp length) that fused hPVextracellular membrane gene with the gene fragment coding human IgG1-Fcfragment was successfully amplified by PCR using the recycled product asa template and the following pair of primers:

Forward primer hPV-1-Fc-F-BglII: (SEQ ID NO: 5)GTGGAGATCTCACGTGAGCACAGAGTCCAACCTG Reverse primer PV1-DHFR-XbaI-R:(SEQ ID NO: 7) TAACTCTAGATCATTTACCCGGGGACAGGG

The recombinant gene DNA amplified by PCR was recycled and treated withendonuclease digestion, cloned into the expression vector pCDNA3.1-DHFRto obtain the recombinant plasmid. The recombinant expression plasmid(name: pQY-DHFR-PV1-Fc) secreting the recombinant gene hPV-1-Fc in CHOcells was proved to be achieved successfully by endonuclease digestionand DNA sequencing identification

1.3 Expression and Preparation of Human PV-1-His Recombinant Protein andPV-1-Fe Fusion Protein (Immunogen)

The above expression plasmids (pQY-DIEFR-PV1-His, pQY-DHFR-PV1-Fc) weremixed with Fugen-6 liposome (Roche) respectively, then transfected intoDHFR gene deficiency CHO cell (CHO-dhfr-). After transfection andscreening by medications (Methotrexate, MTX), the cell lines effectivelyexpressing the human PV-1-His recombinant protein and the human PV-1-Fcfusion protein were obtained. The screened expression cell lines wereamplified and cultured in a serum-free culture medium, then separatedand purified from the cell supernatant using Ni-Affinity chromatographycolumn and Protein-A affinity chromatography column respectively, thehuman PV-1-His recombinant protein and the human PV-1-Fc fusion proteinwith a purity of over 90% were obtained.

FIG. 2 showed the SDS-PAGE electrophoretic analysis of the humanPV-1-His recombinant protein (DTT-reduced). The result showed that themain lanes in the DTT-reduced human PV-1-His protein sample were around55 kd, which was consistent with the theoretical expected molecularweight of the protein.

1.4 A Recombinant Human PV-1 Protein Immunizes Animals

Firstly, the human PV-1-His recombinant protein and Freund's completeadjuvant (Sigma, USA) were mixed, then injected subcutaneously atmultiple points to Balb/c mice (100 μl/mouse, 10 μg PV-1-His proteineach time). After 2-3 weeks of the first immunization, the mixture ofhuman PV-1-Fc fusion protein and Freund's incomplete adjuvant (Sigma,USA) were injected into the mice again subcutaneously at multiplepoints. After 3-4 times of boost immunization, a small amount of themouse serum was collected and tested the titer of anti-PV-1 antibody inthe mouse serum by enzyme-linked immunosorbent assay (ELISA) using a96-well plate coated with the human PV-1-Fc fusion protein. The spleniccells of the mouse with high titer were collected for the cell fusion ofthe next step.

1.5 Cell Fusion

After 3 to 4 days of the last immunization, the splenocytes suspensionof the mouse were prepared in a sterile condition, fused with the mouseSP2/0 myeloma cells (purchased from Cell Center of Shanghai Institute ofLife Sciences, Chinese Academy of Sciences) at a ratio of 5:1 or 10:1under the function of 50% PEG-1000 (Sigma, USA). The cell fusion processfollowed a conventional method (Kohler G and Milstein C: Nature 1975;256:495-497):1 mL PEG was added slowly within 60 seconds, reacted for 90seconds, terminated the reaction with the serum-free RPMI-1640 culturemedium, centrifuged 10 minutes with 1000 rpm, removed the supernatant;the deposited cells under the centrifugal were obtained and adjusted thecells concentration to 1×10⁶/ml with RPM 1640-10% FCS culture mediumcontaining 10% HAT (H for hypoxanthine, A for amino disc poison, forthymidine nucleoside, Sigma, USA), added into 96 well flat cell cultureplate (200 μl/hole), then incubated in an incubator containing 5% CO₂(Thermo, USA) at 37° C. for 2-3 weeks.

1.6 Screening of Mouse Hybridoma Cell with Positive PV-1 AntibodySecretion by immunohistochemistry (IHC) Method

In the example of the present invention, the cell lines with positivePV-1 antibody secretion were screened from the mouse hybridoma cells byImmunohistochemistry (IHC) method.

The process was as follows:

1) CHO cells transfected with the human PV-1 gene (CHO/PV-1) andnon-transfected CHO cells were mixed at a ratio of 1:6 and spread in a96 well cell culture plate, then incubated overnight in an incubatorcontaining 5% CO2 at 37° C.;

2) The cell culture plate was taken out, and the nutrient solution wasabsorbed, fixed with the phosphate buffered saline (PBS) containing 2%paraformaldehyde, permeabilized with 90% methanol.

3) After rinsing with PBS solution, the primary antibody (the mousehybridoma cell supernatant or serum of PV-1 immunized mouse (diluted at1:200) as a positive control sample) was added, incubated at 37° C. for1 hour;

4) After rinsing with PBS solution, the second antibody (HRP-Goatanti-Mouse IgG (1:400)) was added and incubated at 37° C. for 1 hour;

5) After rinsing with PBS solution again, the substrate (DAB, 01% H2O2)was added for staining

FIG. 3 shows the representative results of Immunohistochemistry (IHC)screening.

As shown in FIG. 3, the supernatant of the mouse hybridoma cell culturewith a code name of STW-139-15 (FIG. 3C) can significantly specificallycombine with the mixture of CHO/PV-1 and CHO. The IHC staining intensityand the ratio of positive cells are the same as that of the positivecontrol sample (the serum sample of the Mouse immunized with PV-1antigen, FIG. 3B); the IHC staining results of the supernatant of SP2/0myeloma cell was negative (FIG. 3A), it is also consistent with theexpected results.

EXAMPLE 2 Determining the Binding of the Supernatant Sample of the MouseHybridoma Cell STW-139-15 and the Recombinant Human PV-1-Fe FusionProtein by ELISA

The above primarily screened positive hybridoma cell was diluted to 1-10cells per well with RPMI-1640-10% FCS culture medium, spread in a96-well cell culture plate, incubated in an incubator containing 5% CO₂at 37° C. for 2-3 weeks. After clones grew up, the supernatant wascollected and determined the presence of an anti-PV-1 antibody by ELISA.

The ELBA method was as follows:

1) The 96-well cell culture plate was coated with the recombinant humanPV-1-Fc fusion protein (2 pH 9.6, 0.1 M NaHCO3 solution) at 37° C. for 2hours, 2% Bovine Serum Albumin (BSA) was added and sealed overnight at4° C.

2) The next day, the plate was washed with PBS-0.1% Tween20 solution,followed by the addition of the hybridoma cell culture supernatant to bedetected (an unfused SP2/0 myeloma cell culture supernatant as anegative control) and incubated at 37° C. for 2 hours;

3) After washing with PBS-0.1% Tween20 solution, the HRP-Goat anti-MouseIgG (Sigma, USA) was added and incubated at 37° C. for 1 hour;

4) After washing with PBS-0.1% Tween20 solution again, the substratesolution (OPD, 0.1% H₂O₂) was added for staining about 10-15 minutes;

5) 0.1M HCl solution was added to quench the reaction, then the OD valueat 492 nm was read in Multiskan-FC Microplate Reader (Thermo Scientific,USA).

FIG. 4 is a schematic diagram of the representative results of theELISA.

As shown in FIG. 4, the supernatant sample of the mouse hybridoma cellSTW-139-15 contained high titer antibodies and can specifically bindhuman PV-1-Fc fusion protein, but the supernatant sample of non-relatedantibodies sample mAb113 (anti-SOST antibody, SOSI stands forSclerostin) and SP2/0 myeloma cell were all negative.

EXAMPLE 3 Determining and Analyzing the Binding of the Mouse STW-139-15Monoclonal Antibody and the Human PV-1-Fc Fusion Protein and OtherNon-Related Proteins

In the present example, the binding of the mouse STW-139-15 monoclonalantibody and the human PV-1-Fc fusion protein and other non-relatedproteins was determined by ELISA.

The 96-well ELISA plate was coated with the human PV-1-Fc fusion proteinand other non-related proteins (CD3, TIGIT-His, SIRPa-His) or Fc-fusionprotein (PD1-Fc, PDL1-Fe, PDL2-Fc, mPDL1-Fc, CTLA4-Fc, CD28-Fc, B7-Fcand BTLA-Fc) in the concentration of 1 ug/ml. The mouse STW-139-15monoclonal antibody was added as the primary antibody, followed by theaddition of the HRP-Goat anti-Mouse IgG (Jackson Company) as the secondantibody. After that, the substrate solution (OPD, 0.1% H2O2) was addedfor staining. 1M HCl solution was added to quench the reaction. The ODvalue at 492 nm was read in Multiskan MC Microplate Reader (ThermoScientific, USA).

FIG. 5 showed the ELISA result. The result showed that the murinemonoclonal antibody sample STW-139-15 only specifically bound to thehuman PV-1-Fc fusion protein (OD value>1.0), but did not significantlybind to CD3 and other non-related recombinant proteins (His-labelled, orIgG-Fc fusion protein) (OD value<1.0). The result illustrated thatSTW-139-15 monoclonal antibody has high specificity in antigenrecognition and binding, and only hinds to PV-1 protein.

EXAMPLE 4 Determining and Analyzing the Binding of Murine STW-139-15Monoclonal Antibody and CHO Cell Transfected with Human PV-1 Gene(CHO/PV-1) by Flow Cytometer

In the present example, the murine monoclonal antibody STW-139-15 samplewas used as the primary antibody; the FITC fluorescence-labeled rabbitanti-mouse IgG was used as the second antibody. The binding ofSTW-139-15 monoclonal antibody sample and the CHO cell expressing thehuman PV-1 gene was determined by the flow cytometer.

CHO/PV-1 cell stably transfecting and expressing human full-lengthCHO/PV-1 recombinant protein gene, the supernatant sample of the mousehybridoma STW-139-15, non-related mouse hybridoma mAb21 sample(anti-PD-1 monoclonal antibody), the serum of the mouse immunized withPV-1 antigen (positive control sample, diluted at 1:200) and SP210myeloma cell culture supernatant (negative control) were incubated at 4°C. for 1 hour, rinsed with PBS-0.1% FCS solution, then the FITCfluorescence-labeled rabbit anti-mouse IgG (diluted at 1:200; SouthernBiotech Company) was added and incubated at 4° C. for 1 hour; afterrinsing with PBS-0.1% FCS solution, the samples were tested with BDAccuri C6Plus Flow Cytometer (BD Biosciences, USA).

FIGS. 6A-6D are schematic diagrams of the representative result testedby the flow cytometer. As shown in FIGS. 6C-6D, the supernatant sampleof the mouse hybridoma. STW-139-15, as the same with the positivecontrol sample (FIG. 6C, the serum of the mouse immunized with PV-1protein), significantly binds to CHO/PV-1 cell (FIG. 6D). Instead, thenon-related mouse hybridoma sample (FIG. 6B), the mouse SP2/0 myelomacell culture supernatant as a negative control sample (FIG. 6A) does notspecifically bind to CHO/PV-1 cells.

FIG. 7 showed the antibody's solubility-mean fluorescence curve of aseries of gradient dilutions of murine STW-139-15 monoclonal antibodysample binding with CHO cell stably transfected with human PV-1 gene(CHO/PV-1). It showed that the binding of STW-139-15 monoclonal antibodysample and CHO/PV-1 cell in the solubility range of 0.1-10 ug/mlpresented a dose-response curve.

FIGS. 8A-8C are schematic diagrams of the representative result of themixture sample containing the murine STW-139-15 monoclonal antibodysample, CHO cell, and CHO/PV-1 cell (at a ratio of 9:1) tested by theflow cytometer. As shown in FIGS. 8A-8C, compared with the mouse SP2/0cell supernatant negative control sample (FIG. 8A) and non-relatedhybridoma cell supernatant (FIG. 8B), the murine hybridoma STW-139-15monoclonal antibody sample significantly specifically bind to part ofcells in mixture sample (FIG. 8C). The binding proportion of positivecells was 9.67%; it is consistent with the percentage of CHO/PV-1 cells(10%) in the mixture sample. The result further demonstrated thatSTW-139-15 only specifically recognized and bound to PV-1 antigen; itdid not bind to the other proteins or antigenic substances in CHO cells.

EXAMPLE 5 Determining and Analyzing the Binding of Murine STW-139-15Monoclonal Antibody and Human HUVEC

In the present example, the murine monoclonal antibody STW-139-15 samplewas used as the primary antibody; the FITC fluorescence-labeled goatanti-mouse IgG was used as the second antibody; the binding ofSTW-139-15 monoclonal antibody sample and human HUVE was determined bythe flow cytometer.

HUVEC were permeabilized with 0.1% Triton X-100, followed by theaddition of the mouse hybridoma STW-139-15 supernatant sample or themouse SP2/0 cell supernatant as negative control. Then incubated at 4°C. for an hour and rinsed by PBS-0.1% FCS solution; after that, theFITC-Goat anti-Mouse IgCl (H+L) (Sigma, USA) was added, incubated at 4°C.: for an hour and rinsed by PBS-0.1% FCS solution again. The samplewas tested with BD Accuri C6Plus Flow Cytometer (BD Biosciences, USA).

FIG. 9 is a schematic diagram of the representative result tested by theflow cytometer. As shown in FIG. 9, compared with the mouse SP2/0 cellsupernatant negative control (sample A01 NC), the mouse hybridomaSTW-139-15 cell supernatant sample significantly specifically bound tohuman HUVEC.

EXAMPLE 6 Determining the Binding of the Murine STW-139-15 MonoclonalAntibody and Tissue Sections of Human Normal Tissues byImmunohistochemistry (IHC) Method

In the present example, the binding of the murine STW-139-15 monoclonalantibody sample and tissue sections of part of normal human tissues wasdetermined and analyzed by Immunohistochemistry (IHC) method; thedetection process was as follows:

After rehydration of paraffin sections of normal human tissues andresumption of antigen treatment, the murine monoclonal antibodySTW-139-15 sample was added as the primary antibody, incubated at roomtemperature for 1 hour, and rinsed. Diluted HRP-Goat anti-Mouse IgG(second antibody) was added, incubated at room temperature for 1 hourand rinsed, then the substrate DAB was added for staining, redyed withhematoxylin, the film was sealed and photographed.

FIGS. 10A-10F are schematic diagrams of the representative results ofthe Immunohistochemistry method. As shown in FIGS. 10A-10F, in theimmunohistochemical staining sections of the normal tissue, includinglung, liver, brain, heart, pancreas, and spleen, STW-139-15 monoclonalantibody sample only specifically bound to lung tissue, and the stainingresults with other tissues were not significant. The positiveimmunohistochemistry determination result of STW-139-15 monoclonalantibody in lung tissue was consistent with the expression result inlung tissue reported in the literature. The cDNA coding PV-1 antigen wasinitially separated and cloned from rat lung tissue (Stan R V et al.,1999 J Cell Biol. 145:1189-98).

EXAMPLE 7 Determining the Binding of Murine STW-139-15 MonoclonalAntibody and Tissue Sections of Human Tumor Tissues byImmunohistochemistry (MC) Method

In the present example, the binding of murine STW-139-15 monoclonalantibody and tissue sections of partial human tumor tissues wasdetermined and analyzed by Immunohistochemistry (IHC) method; thedetection process was as follows:

After rehydration of paraffin sections of human tumor tissues andresumption of antigen treatment, the murine monoclonal antibodySTW-139-15 sample was added as the primary antibody, incubated at roomtemperature for 1 hour and rinsed, diluted HRP-Goat anti-Mouse IgG(second antibody) was added, incubated at room temperature for 1 hourand rinsed, the substrate DAB was added for staining, redyed withhematoxylin, the film was sealed and photographed.

FIGS. 11A-11F are schematic diagrams of the representative results ofthe Immunohistochemistry method. As shown in FIGS. 11A-11F, STW-139-15monoclonal antibody specifically bound to vascular-like structure invarious tumor tissues (including lung cancer, liver cancer, brain tumor,pancreatic cancer, ovarian cancer, etc.). However, the staining resultwith the lymphoma tissue section was not significant.

Based on the fact that STW-139-15 monoclonal antibody specifically boundto various tumor tissues and did not bind to most normal tissues (seethe result of Example 6), this monoclonal antibody should be the idealsubstance or carrier for preparing the medication or formulationtargeting blood vessels of tumor region.

EXAMPLE 8 Determining the Binding of Murine STW-139-15 MonoclonalAntibody and Macaca Fascicularis PV-1. Protein by Flow Cytometer

1) Amino Acid Sequences Comparison and Analysis of PV-1 ProteinExtracellular Membrane Region of the Human and Monkey

The comparison and analysis result of amino acid sequences of human PV-1protein extracellular membrane region (SEQ ID NO: 8) and amino acidsequences of Macaca Fascicularis PV-1 protein extracellular membraneregion of) extracellular membrane region (SEQ ID NO: 25) was shown inFIG. 12. As shown in FIG. 12, there are 95% homology in proteinsequences between the extracellular membrane region of MacacaFascicularis PV-1 protein and the extracellular membrane region of humanPV-1 protein; there are 17 amino acid difference sites.

2) Construction of CHO Cell Line Expressing Monkey PV-1 Gene

According to amino acid sequences of Macaca Fascicularis PV-1full-length protein published in Genbank (NCBI: GenBank: AKG92647.1),the responding cDNA fragment of Macaca Fascicularis PV-1 was delegatedto Suzhou Genewiz Biological Technology Co. LTD to artifitiallysynthesize, after that treated with the restriction DNA endonuclease,cloned into the expression plasmid pCDNA3.1-DHFR, then the recombinantplasmid was obtained. After treating with restriction endonucleasedigestion and DNA sequencing and identification, the recombinant plasmidexpressing Macaca Fascicularis PV-1 full-length protein in CHO-dhfr cellmembrane (Plasmid name: pCDNA3.1-DHFR-mkPV1) was successfully obtained.

The above-expressed plasmid DNA was mixed with Fugen-6 liposome (Roche),then transfected into DHFR gene deficiency CHO cell (CHO-dhfr-). Aftertransfection, screened by regular IMDM culture medium containing 8% FBS,the cell line expressing Macaca. Fascicularis PV-1 protein was obtained.

3) Analyzing the Binding of the Murine STW-139-15 Monoclonal Antibodyand CHO/Monkey PV-1 Cell by Flow Cytometer

The binding of the murine STW-139-15 monoclonal antibody sample and theabove CHO cell expressing Macaca Fascicularis PV-1 full-length protein(CHO/Monkey PV-1) was determined and analyzed by the flow cytometermethod as described in Example 4. The representative detection result ofthe flow cytometer was shown in FIGS. 13A-13C, compared with thenegative control sample (SP2/0 myeloma cell culture supernatant, FIG.13A) and non-related mouse monoclonal antibody mAB7 sample (anti-PD-1monoclonal antibody, FIG. 13B), the murine monoclonal antibodySTW-139-15 significantly bound to CHO/Monkey PV-1 cell (FIG. 13C). Theresult primarily demonstrated that the different sites between aminoacid sequences of Macaca Fascicularis PV-1 protein and amino acidsequences of human PV-1 protein did not affect the binding of STW-139-15monoclonal antibody and CHO/Monkey PV-1 cell. The result of STW-39-15monoclonal antibody and CHO/Macaca Fascicularis PV-1 cell also suggestedthat Macaca Fascicularis is the ideal and related animal for studyingSTW-139-15 monoclonal antibody.

EXAMPLE 9 Cloning, Amplification, and Analysis of the Genes Coding theVariable Regions of the Murine STW-139-15 Monoclonal Antobody

In the present example, the total RNA was extracted from the mousehybridoma cell STW-139-15, and used as a template; together with thedegenerate primers, to clone and amplify the cDNA gene fragments ofSTW-139-15 antibody heavy chain variable region and light chain variableregion respectively by reverse transcription-polymerase chain reaction(RT-PCR) method (Wang Y et al : Degenerated primer design to amplify theheavy chain variable region from immunoglobulin cDNA, BMCBioinformatics. 2006; 7 Suppl (4): S9). Wherein the cDNA gene cloningprocess was as follows:

Step 1: The total RNA was extracted from the mouse hybridoma cellSTW-139-15 by RNA extraction reagent (RNAi so Plus, Takara Company)

Step 2: cDNA template was obtained in Eppendorf tube by RT-PCR method

Wherein, the primer's sequence of the reverse transcription-polymerasechain reaction for STW-139-15 antibody light chain variable region(STW-139-15-L) was TGT CGT TCA CTG CCA ICA AT (SEQ ID NO: 9)

The primer's sequence of the reverse transcription-polymerase chainreaction for STW-139-15 antibody heavy chain variable region(STW-139-15-L) was GCA AGG CIT ACA ACC ACA (SEQ ID NO: 10):

RT-PCR reaction system was as follows:

Primer  2 μl RNA template 30 μl Incubated at 72° C. for 10 minutes, thenstayed on ice for 2 minutes

Followed by

5 × RT-PCR reaction buffer  10 μl dNTPs   5 μl PrimeScript reversetranscription-polymerase 1.5 μl Distilled water 1.5 μl Total volume  50μl

Reacted at 42° C. for 1 hour, then increased to 75° C., after 15minutes, inactivated, the cDNA was obtained and stored at −20° C. forlater use.

Step 3: PCR cloning and amplification of STW 39-15 an body light chainvariable region gene and heavy chain variable region gene.

The following pair of primers used in cloning and amplification ofSTW-139-15 antibody light chain variable region gene by degenerateprimers PCR method were as follows:

Forward primer: (SEQ ID NO: 11) GAC ATT GTG ATG WCM CA Reverse primer:(SEQ ID NO: 12) CTG AGG CAC CTC CAG ATG TTwherein W = A or T, M = A or C.

The following pair of primers used in cloning and amplification ofSTW-139-15 antibody heavy chain variable region gene by degenerateprimers PCR method were as follows:

Forward primer: (SEQ ID NO: 13) CAR CTG CAR CAR YCT GWherein, R = A or G, Y = C or T. Reverse primer: (SEQ ID NO: 14)GTG CTG GAG GGG ACA GTC ACT

DNA products amplified by PCR were analyzed by electrophoresis in 1%agarose gel. When electrophoresis is over, the separated bands were cutand sequenced to obtain the nucleotide sequences of the antibody's lightand heavy chain variable region DNA. The nucleotide sequence of thelight chain variable region DNA was set forth in SEQ ID NO: 15. Theamino acid sequence of the light chain variable region DNA inferred fromthe DNA nucleotide sequence was set forth in SEQ ID NO:16. The aminoacid sequences of CDR1, CDR2, and CDR3 of the light chain antigencomplementarity-determining regions (CDR) were set forth in SEQ ID NO.:17, SEQ ID NO.: 18, and SEQ ID NO.: 19, respectively.

The nucleotide sequence of the heavy chain variable region DNA was setforth in SEQ ID NO: 20, and the amino acid sequence of the heavy chainvariable region DNA inferred from the DNA nucleotide sequence was setforth in SEQ ITS NO: 21. The amino acid sequences of CDR1, CDR2, andCDR3 of the heavy chain antigen complementarity-determining regions(CDR) were set forth in SEMI ID NO.: 22, SEQ ID NO.: 23 and SEQ ID NO.:24, respectively.

EXAMPLE 10 Construction of Human-Mouse Chimeric Antibody STW-139-15-C

The murine STW-139-15 antibody light and heavy chain variable regiongenes obtained by cloning and amplification in Example 9 were fusedseparately with a human kappa light chain constant region (C-domain) anda human IgG1 heavy chain constant region gene fragment to obtain thehuman-mouse chimeric light chain gene (STW-139-15-L) and the human-mousechimeric heavy chain gene (STW-139-15-H). After that, the light, andheavy chain chimeric genes were separately cloned into the expressionplasmid pcDNA3.1 (Invitrogen), followed by transferring into E. Coli toamplify, and separate, then the expression plasmids containing thehuman-mouse chimeric light chain gene and the human-mouse chimeric heavychain gene were obtained.

After that, the partial expression plasmid samples containing thehuman-mouse chimeric light chain gene (recombinant plasmid code: L17,L18, and L19) and the partial expression plasmid samples containing thehuman-mouse chimeric heavy chain gene (recombinant plasmid code: H12,H13, and H15) were combined in pair respectively, mixed with liposome(Roche) and transfected into CHO cell. After 2 to 3 days of cellstransfection, the culture supernatant was collected. The 96-well coatedwith human PV-1-Fc fusion protein, HRP-Goat anti-Mouse IgG (FabSpecific) as the second antibody (Purchased from Shanghai Xitang Biologycompany), the second tested antibody, was used to read the value at 492nm in Microplate Reader to detect the binding of the chimeric antibodyand human PV-1 protein.

The ELISA representative result was shown in the following Table 1 andFIG. 14:

TABLE 1 Analyzing the binding activity of the transient transfected cellculture supernatant and human PV1-Fc protein by ELISA method DilutionTimes 2 4 8 16 32 64 128 256 512 1024 2048 4096 Light chain and H12 +L17 0.057 0.056 0.057 0.053 0.050 0.055 0.051 0.073 0.054 0.054 0.0520.055 heavy chain H13 + L18 0.055 0.065 0.054 0.060 0.054 0.052 0.0520.053 0.055 0.051 0.053 0.054 transfection H15 + L19 0.214 0.134 0.0960.100 0.054 0.059 0.061 0.055 0.054 0.052 0.053 0.055 samples (Note:L17, L19 are light chains with correct sequences; H15 is a heavy chainwith correct sequences; H12, H13 and L18 are chains with wrongsequencing results)

As shown in Table 1 and FIG. 14, the CHO cell culture supernatanttransfected with correctly expressed human-mouse chimeric antibodySTW-139-15 heavy chain gene plasmid and correctly expressed light chaingene plasmid (HIS+L19) can specifically bind to human PV-1-Fc protein.

The above-transfected cell culture supernatant was centrifugated andfiltered with a 0.45 μm filter membrane. It was loaded to a Protein-Achromatography affinity column (Protein-A Sepharose Fast Flow; GE, USA)and purified to obtain the human-mouse chimeric antibody (STW-1394-15-C)with a purity of over 95%.

Purified STW-139-15-C antibody protein was sterilized, then dissolved insterile. PBS solution to prepare the liquid formulation with a finalprotein solubility of around 10 mg/ml, which can be stored at a lowtemperature of 2-8° C. away from light for a long time.

EXAMPLE 11 Determining the Biological Efficacy or Activity of theHoman-Mouse Chimeric Antibody (STW-139-15-C) In Macaca Fascicularis

STW-139-15 does not recognize the mouse PV-1, so its biological efficacyor activity can not be tested in the mouse. Therefore, in the presentexample, Macaca Fascicularis was chosen as test animals to determine invivo the effect of human-mouse chimeric antibody STW-139-15-C on theinhibition of choroidal neovasculature in Macaca Fascicularis induced bylaser. The study was delegated to Chengdu Westchina-Frontier PharmaTechCo., (WCFP) and the National Chengdu New Drug Safety Evaluation Centerto complete.

Objective

Study the effects of human-mouse chimeric antibody (STW-139-15C, samplecode: STW007) through vitreous injection on choroidal neovascularizationleakage and growth induced by laser in Macaca Fascicularis and providean animal experimental basis for further study of this drug.

The present aminal experimental study was divided into two stages,wherein the experimental model, administration grouping, andexperimental results in the first stage are described as follows:

Experimental Model and Administration Grouping

11.1 Modeling

11.1.1 Anesthetizing

Macaca Fascicularis were anesthetized with pentobarbital sodium (25mg/kg, intravenous injection), and a small amount of Refresh Celluvisc(Carboxymethylcellulose Sodium) was added irregularly during anesthesiato keep the cornea moist.

11:1.2 Dilating Pupils

Mydrin-P (compound tropicamide eye drops) was applied to both eyes todilate pupils.

11.1.3 Laser Photocoagulation

The head of Macaca Fascicularis was fixed in front of the ophthalmiclaser photocoagulation, and the macular area was photocoagulated byretinoscope. Photocoagulation around macular fovea but avoid damage tofovea, irradiation 8-9 points per eye. Laser parameters: spot diameter50 μm, energy 0.6-0.7 W, exposure time 0.05 s. Determination ofsuccessful photocoagulation: bubbles can be seen to indicate thatBruch's membrane was broken.

One Fluorescein angiography was performed during 2 to 3 weeks afterlaser photocoagulation to judge the success of the modeling.

The Macaca Fascicularis had at least one light spot of grade 4 on eacheyeball to judge the success of the modeling.

11.2 Dosage Design

The animals in each group were administered in the third week afterlaser photocoagulation. The dosage design was shown in Table 2:

TABLE 2 Dosage Design Dosage of Drug Drug Number Group MaterialAdministration Administration Concentration volume of Description TestedRoute mg/eye mg/mL μL/eye Animals Model 0.9%NaCl Vitreous — — 50 1control Injection injection group Positive Positive Drug Vitreous 0.5 2025 1 control hPV19 injection group monoclonal antibody STW007 STW007Vitreous 0.5 10 50 1 monoclonal injection antibody (STW- 139-15C)STW007 + STW007 Vitreous 0.25 10 25 1 Positive monoclonal injection druggroup antibody (STW- 139-15C) Positive Drug 0.25 20 12.5 hPV19monoclonal antibody

The positive drug hPV19 monoclonal antibody was a humanized antibodyspecifically recognizing and binding human and monkey VEGF antigen (SeeChinese patent document, ZL: 201210540692X, patent name: Monoclonalantibody for antagonizing and inhibiting binding of vascular endothelialgrowth factor to its receptor, and coding sequence; and the UnitedStates patent document: U.S. Pat. No. 9,580,498B2)

11.3 Administration

Administration route: vitreous injection in both eyes;

The reason for administration route: consistent with the clinicaladministration route;

Administration frequency: single dose;

Drug delivery method: each group of Macaca Fascicularis was anesthetizedwith pentobarbital sodium (around 25 mg/kg, intravenous injection,appropriate adjustments can be done according to the monkey anesthesiasituation), disinfected the eyes to be injected with povidone-iodinesolution. Table 2 showed that the corresponding concentration of STW007,positive drug, STW007 and positive drug were injected by vitreousinjection in both eyes; the model control group was administered 0.9%NaCl injection with the same volume. If necessary, 1 to 2 drops ofOxybuprocaine Hydrochloride eye drops should be dropped into the eyes tobe injected to conduct the surface anesthesia, then injected.

After vitreous injection, 1 to 2 drops Ofloxacin eye cream was droppedto resist infection and moisten the cornea.

The day of administration is defined as the first day of the trial.

The Second Stage: Animal Experiments Results

The effects of vitreous injection of STW-139-15C monoclonal antibody(STW007) and positive control drug hPV19 (anti-VEGF monoclonal antibody)on the reduction of fluorescein leakage area and the improvement rate ofMacaca Fascicularis on the third week after photocoagulation were shownin Table 3 (statistical data up to the 49th day after administration).

FIG. 15A to FIG. 15D showed fundus fluorescein images of each group at7, 14, and 21 days after vitreous injection.

TABLE 3 Effects of vitreous injection of STW007 on the reduction offluorescein leakage area and the improvement rate of Macaca FascicularisSample group 1 + 2 Model control Sample group 1 Sample group 2STW139-15C group 0.9% NaCl STW-139-15C anti-VEGF Mab and Index/Injection Mab hPV19 hPV19Mab Time Determined n X ± SD n X ± SD n X ± SDn X ± SD Reduction of fluorescein leakage area (mm²) 7 days after 2−22.347 ± 0.347 2 19.749 ± 5.455 2 12.075 ± 14.310 1 25.866administration 14 days after 2 −21.091 ± 7.675 2  13.133 ± 12.150 212.877 ± 14.641 1 25.560 administration 21 days after 2 −15.778 ± 1.9082 16.129 ± 9.182 2 14.207 ± 16.845 1 25.975 administration 25 days after2  −3.288 ± 8.222 2  17.536 ± 10.119 2 14.377 ± 16311  1 27.733administration 36 days after 2  −1.537 ± 25.918 2 21.483 ± 4.933 215.541 ± 20.343 1 23.387 administration 42 days after 2  −2.837 ± 19.8082 23.191 ± 8.579 2 17.663 ± 20.082 2 30.934 ± 6.034 administration 49days after 2  −10.161 ± 20.181 2 17.011 ± 4.580 2 16.672 ± 18.410 226.167 ± 9.461 administration Improvement rate of fluorescein leakagearea (%) 7 days after 2  −54.73 ± 17.64 2 55.36 ± 0.53 2 45.75 ± 18.72 188.79  administration 14 days after 2 −48.65 ± 2.91 2  33.27 ± 24.52 251.84 ± 13.99 1 87.74  administration 21 days after 2  −39.27 ± 16.51 2 43.24 ± 13.36 2 53.78 ± 22.10 1 89.17  administration 25 days after 2 −11.12 ± 22.56 2  46.96 ± 14.93 2 58.06 ± 15.27 1 95.20  administration36 days after 2  −13.49 ± 64.48 2 60.64 ± 3.50 2 49.37 ± 42.64 1 80.28 administration 42 days after 2  −14.37 ± 50.53 2 64.10 ± 5.72 2 71.12 ±19.17 2 93.85 ± 3.27 administration 49 days after 2  −32.41 ± 56.94 247.73 ± 0.81 2 69.82 ± 12.84 2  78.31 ± 16.19 administration Note: Atthe time points from day 7 to day 36 after administration, there were nodata on the leakage area in sample group 1 + 2 (4M001) on the right eye,so the sample size was 1.

As shown in Table 3 and FIG. 15A-15D, compared with beforeadministration, fundus fluorescein leakage in the model control group(0.9% NaCl injection) after administration showed no improvement butincreased (the average improvement rate of fluorescein leakage areaduring the observation period was −11.12%˜54.73%; fundus fluoresceinseries images from day 7 to day 21 were shown in FIG. 15A). On the otherhand, the fundus fluorescein leakage of STW-139-15C monoclonal antibodysample (0.5 mg/eye) was significantly improved from day 7 afteradministration (the improvement rate of fluorescein leakage area duringthe observation period was 33.27%˜64.10%). Fundus fluorescein seriesimages from day 7 to day 21 were shown in FIG. 15B) the reduction offluorescence leakage was similar to the results of the positive controldrug hPV19 (anti-VEGF nab) group at the same dose (0.5 mg/eye) (theimprovement rate of fluorescein leakage area during the observationperiod ranged from 45.75% to 71.12%, and fundus fluorescein seriesimages from day 7 to day 21 were shown in FIG. 15C).

After adminitering STW-139-5C monoclonal antibody sample (0.25 mg/eyes)in combination with the positive control drug hPV19 monoclonal antibody(0.25 mg/eyes), fundus fluorescein leakage area during the observationperiod maintained between 80.28%˜95.20% (fundus fluorescence angiographyseries images from day 7 to day 21 after administration were shown inFIG. 15D). Under the condition of halving the injection dose of a singledrug, its effect is better than two times the dose of positive controldrug hPV19 monoclonal antibody (0.5 mg/eyes). The result showed thatSTW-139-15C monoclonal antibody specifically binding to PLVAP/PV-1 had asignificant inhibitory effect on laser-induced choroidalneovascularization in Macaca Fascicularis. When STW-139-15C monoclonalantibody and VEGF monoclonal antibody were combined, the efficacy wasbetter than each single drug treatment.

1. A monoclonal antibody or a derivative thereof specifically binding tohuman plasmalemma vesicle-associated protein, comprising a firstvariable region and a second variable region, wherein the first variableregion is an antibody light chain variable region comprising antigencomplementarity-determining regions CDR1, CDR2 and CDR3 having aminoacid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ IDNO: 19, respectively; and wherein the second variable region is anantibody heavy chain variable region comprising antigen complementaritydetermining regions CDR1, CDR2 and CDR3 having amino acid sequences asset forth in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24,respectively.
 2. The monoclonal antibody or the derivative thereofaccording to claim 1, wherein the first variable region is an antibodylight chain variable region having an amino acid sequence as set forthin SEQ ID NO: 16; and the second variable region is an antibody heavychain variable region having an amino acid sequence as set forth in SEQID NO:
 21. 3. The monoclonal antibody or the derivative thereofaccording to claim 1, comprising the antibody light chain variableregion, a human antibody light chain constant region, the antibody heavychain variable region, and a hinge region of a human antibody heavychain constant region, a CH1 region, a CH2 region, and a CH3 region. 4.The monoclonal antibody or the derivative thereof according to claim 3,wherein the human antibody light chain constant region is a kappa chainor a lambda chain of a human antibody, the human antibody heavy chainconstant region is a human IgG-1 isotype, a human IgG2 isotype, a humanIgG3 isotype, a human IgG4 isotype, a human IgA, or a human IgM.
 5. Themonoclonal antibody or the derivative thereof according to claim 1,wherein the derivative is a Fab fragment of an antibody, an Fv fragment,a single-chain antibody, a hi-specific antibody, an antibody-drugconjugate, or a chimeric antigen receptor T-cell.
 6. A DNA molecule or agene encoding the monoclonal antibody or the derivative thereofaccording to claim 2, wherein a nucleotide sequence encoding theantibody light chain variable region is set forth in SEQ ID NO: 15, anucleotide sequence encoding the antibody heavy chain variable region isset forth in SEQ ID NO:
 20. 7. An expression vector comprising a DNAsequence of the DNA molecule of claim 6 and an expression regulatorysequence operably linked to the DNA sequence.
 8. A recombinant hostcell, wherein the recombinant host cell is transfected with theexpression vector of claim
 7. 9. The recombinant host cell according toclaim 8, wherein the recombinant host cell or a progeny cell thereofexpresses a monoclonal antibody or a derivative thereof, wherein themonoclonal antibody or the derivative thereof comprises a first variableregion and a second variable region, wherein the first variable regionis an antibody light chain variable region comprising antigencomplementarity-determining regions CDR1 CDR2 and CDR3 having amino acidsequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:19, respectively and wherein the second variable region is an antibodyheavy chain variable region comprising antigencomplementarity-determining regions CDR1, CDR2 and CDR3 having aminoacid sequences as set forth in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ IDNO: 24, respectively.
 10. A pharmaceutical composition, comprising apharmaceutically effective amount of the monoclonal antibody or thederivative thereof of claim 1, and a pharmaceutically accepted carrierthereof.
 11. The pharmaceutical composition according to claim 10,wherein the pharmaceutical composition further comprises apharmaceutically effective amount of an active component antagonizingand blocking VEGF or VEGF R, and a pharmaceutically accepted carrierthereof.
 12. A method of using pharmaceutical composition according toclaim 10, comprising a step of using the pharmaceutical composition fora preparation of a medicament for a treatment of an angiogenesis orosmosis-related disease.
 13. The method according to claim 12, whereinthe angiogenesis or osmosis-related disease is a choroidalneovascularization fundus disease.
 14. The method according to claim 13,wherein the choroidal neovascularization fundus disease is diabeticretinopathy and age-related macular degeneration.
 15. A monoclonalantibody or a derivative thereof binding to plasmalemmavesicle-associated protein of both human and monkey, wherein themonoclonal antibody or the derivative thereof binds to antigens havingan amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 25,and competitively hinds to PV-1 in presence of the monoclonal antibodyor the derivative thereof of claim
 1. 16. A method of antagonizing andblocking angiogenesis or osmosis in vivo mediated by plasmalemmavesicle-associated protein, comprising a step of administering anappropriate amount ©f the monoclonal antibody or the derivative thereofof claim
 1. 17. A reagent or a kit for detecting plasmalemmavesicle-associated protein in a tissue sample or a cell sample,comprising the monoclonal antibody or the derivative thereof of claim 2.18. A method for preparing the monoclonal antibody or the derivativethereof of claim 2, comprising the following steps: a) providing anexpression vector, wherein the expression vector comprises a DNAsequence encoding the monoclonal antibody or the derivative thereof, andan expression regulatory sequence operably linked to the DNA sequence,wherein a nucleotide sequence encoding the antibody light chain variableregion is set forth in SEQ ID NO: 15, and a nucleotide sequence encodingthe antibody heavy chain variable region is set forth in SEQ ID NO: 20;b) transfecting a host cell with the expression vector of step a); c)culturing the host cell from step b) under conditions suitable for anexpression of the monoclonal antibody or the derivative thereof; andisolating, purifying, and collecting the monoclonal antibody or thederivative thereof from a host cell culture medium by affinitychromatography.
 19. The monoclonal antibody or the derivative thereofaccording to claim 2, comprising the antibody light chain variableregion, a human antibody light chain constant region, the antibody heavychain variable region, and a hinge region of a human antibody heavychain constant region, a CH1 region, a CH2 region, and a CH3 region. 20.The monoclonal antibody or the derivative thereof according to claim 2,wherein the derivative is a Fab fragment of an antibody, an Fv fragment,a single-chain antibody, a bi-specific antibody, an antibody-drugconjugate, or a chimeric antigen receptor T-cell.